very easy sticky end ligation: no colonies! - Cloning masters, please help doomed cloner! (Dec/17/2006 )
if your vector digestion release a small fragment that can be lost during column based purification : Go for it and avoid gel purification.
Considering your pcr fragments and the vector :
i think that if you gel purify and do a phenol chlo, it would be nice not to inactivate enzymes.
I consider that such a preparation will be of a very good quality and so inactivation is just here for altering quality
thank you everyone for your suggestions!
so i have troubleshooted ligation mix, since i froze and thaw it three times before eliqouting it.
I have cut my vector (that contains another insert as we recieved it from someone, so i have to cut it out from the gel) and LMT agarose purified, followed by phenol/chloroform and then isopropanol precipitation. (Fred, I didnt inactivate the enzymes here, thanx for the tip)
I took 1 ul of that preparation and incubated with the quick ligase 4 different samples: 5 min, 30 min, 1 hour, and 2 hours. I electroporate and plate .......no colonies .
Can I conclude that my ligation mix is dead since i froze and thaw it several times, before realizing that I shouldn't ? What should I do now?
ok my freind here suggested i prepare my own buffer. So it is T4 DNA ligase from ROCHE and the kit has two buffers DNA dilution buffer and DNA ligation buffer, the ingredients are not written anywhere. Should I prepare both? Just search for some recipe on the internet? by the way the kit was bought in 2002 do you think it is too old to use?
depends... has it been used since that time?
Ligase goes off rather quickly if subjected to freeze thaw cycles. (2- 3 wouldn't matter, but 10+ and you'll start seeing things happening)
Personally I wouldn't use it. If the experiment isn't going well, you don't want the worry of "Is my ligase working". It is just one worry more. Buy new T4 ligase. NEB, Roche, I think anyone would do.
i just noticed after re-reading the protocol that i should have diluted DNA dilution buffer to 1X and i was using it directly (5X)
It is not written anywhere what does this buffer contain maybe 5x was inhibiting my ligation
but also it is written that ligation mix should be purified with some PCR purification kit. my professor says "you it directly without purification", maybe it also has some effect.
well, i guess i should try diluting the buffer and try again. i am so tired of this!!!!
actually i had to find new version of the protocol where this step is written ahead of the procedure. in my old version it is not written clearly. maybe many poeple did same mistake and then they decided to write it in the upper box and in bold
expiry date is not written on the kit, but the only date is written is march 2002. I wonder if this is expiry date?
ok i just ran the gel with undigested and linear vector and it seems to ligate fine in both 5X and 1X DNA dilution buffer the supercoil forms are a little different. i will electroporate it to see....
update: no colonies today too . so what is happening now? i can see that the vector is ligated on the gel, but i get no colonies on the plate. the electrocompetant cells are fine. vector concentration seems fine too. WHAT IS IT???? please help i feel like im losing my mind over this!!!!!!!!!!!!
could you clarify;
which plasmid was transformed?
both the uncut plasmid and the digested plasmid (cut with Xba and EcoRI?)?
If the uncut plasmid is also failing to transform and you're seeing ligation products in the
-there might be problems with the ligase buffer. Nearly all quick ligases buffers use PEG, which I am told is inhibitory to transformation. Ethanol percipitate the DNA. PEG is soluble in EtOH.
-E coli is sensitive to detergent. And detergent can get into your ligation mix from recycled electroporation cuvettes which have not been cleaned properly, some restriction buffers contain small amount of detergent.
-the selection marker on the plasmid is wrong, ie plating on the wrong type of selection.
sorry not to be clear: i wanted to just trouble the ligation step, so I cut the vector with only EcoRI and then purify and then try to religate it. very simple very easy.
I didnt try to ethanol precipitate but i ethanol precipitated all the plasmids that were not working before. it made no difference.
I am also thinking about the plates. However i am sure that i have put kanamycin and plated kanamycin resistant plasmid.
If it is the problem with the ligation buffer then how do I see that it is ligated on the gel???
I am just tired of repeating same steps again and again. i really want to know why my cloning are not working?!?!?
this is rather alarming.
a EcoRI cut plasmid, self ligated and transformed. Ligated DNA is seen on gel.... resulting is no colonies
If you do see the vector recircularising (could you post the picture?), then it would seem that there is no error in the ligation.
So the error much be in the transformation. DNA, protocol or cells
-Has you DNA been exposed to UV light? Excessive exposured to UV (mins) will drop the ligation efficiency a minimum of 10 fold....the longer the exposure, the lower the efficiency. Ask yourself how long has your DNA been under UV light?
Nowadays, I avoid exposing my DNA to UV. I cut around the area where the DNA could have run, leaving a frame. I make that frame a little wider so to include a bit of the running area. I keep the gel slab and I expose the frame to UV. The bit of the running area on the gel frame, shows where the DNA band that I want is. (AS you would expect this bit of DNA is sacrificed for visualisation.) I make indentations on either side of the frame, indicating where my band is. Then I put the gel slab and frame back together. I use the indentation to cut out a gel band, whcih should contain my DNA fragment.
-Could your competent cells have died?
Well not really, but sometimes with home made competent cells, there are low efficiency batches. Could somebody have left out the competent cells, allowing them to defrost... then put them back again?
-Is it possible to get on site help from another PhD student of post-doc?